Bacterial DNA is a potent immunomodulatory substance. Yamamoto S et al., Microbiol Immunol 36:983-997 (1992). It has been hypothesized to be a pathogen-derived ligand recognized by an unidentified pathogen recognition receptor that initiates a host of innate and adaptive immune responses. Wagner H, Adv Immunol 73:329-368 (1999). CpG motif-containing oligodeoxynucleotides (CpG ODN) can mimic the biology of bacterial DNA. Krieg A M et al., Nature 374:546-549 (1995). CpG ODN and DNA vectors have recently been shown to be of clinical value due to immunostimulatory, hematopoietic and adjuvant qualities.
The adaptive immune system appeared approximately 450 million years ago when a transposon that carried the forerunners of the recombinase activating genes, RAG-1 and RAG-2, was inserted into the germ line of early jawed vertebrates. Agarwal A. et al., Nature 394:744 (1998). The ability to mount an adaptive immune response allowed organisms to remember the pathogens that they had already encountered, and natural selection made the adaptive immune response a virtually universal characteristic of vertebrates. However, this did not lead to discarding the previous form of host defense, the innate immune system. Indeed, this earlier form of host defense has been coopted to serve a second function, stimulating and orienting the primary adaptive immune response by controlling the expression of costimulatory molecules.
It had been surmised for a decade that cells of the innate immune system bear receptors for conserved molecular patterns associated with microbial pathogens. According to this model, when the protein antigens derived from pathogens are processed and presented as peptides that serve as the stimulus for specific T cell receptors, pattern recognition receptors (PRRs) on the antigen-presenting cells also induce the synthesis of costimulatory molecules, cytokines, and chemokines. These activated antigen-presenting cells serve to attract and activate the antigen-specific T cells that are essential to all adaptive immune responses. Janeway C A J, Cold Spring Harbor Symp Quant Biol 54:1 (1989); Fearon D T et al., Science 272:50 (1996); and Medzhitov R et al., Cell 91:295 (1997). It was known that the substances that can induce costimulation include bacterial lipopolysaccharide (LPS), synthetic double-stranded RNA, glycans, and mannans. Furthermore, experimental evidence indicated that the processed antigen ligand for the T cell had to be on the same cell as the costimulatory molecule. This is obviously of crucial importance for maintaining self-tolerance; bystander presentation of costimulatory molecules would mean that tolerance would be lost whenever an infection occurred.
To validate this model, it was necessary to identify receptors for microbial patterns that, upon binding pathogen ligands, initiate signaling cascades leading to the production of costimulatory molecules and cytokines. Molecules such as mannose binding protein (MBP) do not qualify for this role, because they activate proteolytic cascades or promote phagocytosis but are not known to induce costimulation. The break-through came with the identification of a human homologue of Drosophila Toll initially cloned as a cDNA and later named hTLR4 (for human Toll-like receptor). Medzhitov R et al., Nature 388:394 (1997); Rock F L et al., Proc Natl Acad Sci USA 95:588 (1998); Chaudhary P M et al., Blood 91:4020-4027 (1998).
Toll-like receptors (TLRs) are a family of germline-encoded transmembrane proteins that facilitate pathogen recognition and activation of the innate immune system. Hoffmann J A et al., Science 284, 1313-1318 (1999); Rock F L et al., Proc Natl Acad Sci USA 95:588-593 (1998). TLRs engage conserved pathogen-derived ligands and subsequently activate the TLR/IL-1R signal transduction pathway to induce a variety of effector genes. Medzhitov R et al., Mol Cell 2:253-258 (1998); Muzio M et al., J Exp Med 187:2097-2101 (1998).
So far, ten different mammalian TLRs have been described. Rock F L et al., Proc Natl Acad Sci USA 95:588-593 (1998); Chaudhary P M et al., Blood 91:4020-4027 (1998); Takeuchi O et al., Gene 231:59-65 (1999); Aderem A. et al., Nature 406:782-7 (2000). So far, genetic data suggest that the TLRs have unique functions and are not redundant. Ligands for and the function of most of these TLRs, aside from TLR2 and TLR4, remain to be elucidated.
It turns out that an LPS-binding and signaling receptor complex is assembled when hTLR4 interacts with LPS bound to CD14, a peripheral membrane protein held to the cell surface by a glycosyl-phosphoinositol tail. The presence of LPS binding protein (LBP) further increases signaling. The hTLR4 protein has a leucine-rich repeat sequence in its extracellular domain that interacts with CD14 complexed with LPS. TLR4 then transduces the LPS signal across the membrane because destructive mutation of this gene lead to an LPS-unresponsive state in mice, which are also deficient in the clearance of Gram-negative bacteria. Poltorak A et al., Science 282:2085 (1998); Qureshi S T et al., J Exp Med 189:615-625 (1999); Eden C S et al., J Immunol 140:180 (1988). It has since become apparent that humans, like flies, have numerous Toll-like receptors (TLRs).
TLR4 and other TLRs have a cytoplasmic Toll/IL-1 receptor (TIR) homology domain. This domain communicates with a similar domain on an adapter protein (MyD88) that interacts with TLR4 by means of a like:like interaction of TIR domains. The next interaction is between the adapter and a kinase, through their respective “death domains.” The kinase in turn interacts with tumor necrosis factor (TNF) receptor-associated factor-6 (TRAF6). Medzhitov R et al., Mol Cell 2:253 (1998); Kopp E B et al., Curr Opin Immunol 11:15 (1999). After TRAF6, two sequential kinase activation steps lead to phosphorylation of the inhibitory protein IκB and its dissociation from NF-κB. The first kinase is a mitogen-activated kinase kinase kinase (MAPKKK) known as NIK, for NF-κB-inducing kinase. The target of this kinase is another kinase made up of two chains, called IκB kinase α (IKKα) and IκB kinase β (IKKβ), that together form a heterodimer of IKKα:IKKβ, which phosphorylates IKβ. NF-κB translocates to the nucleus to activate genes with κB binding sites in their promoters and enhancers such as the genes encoding interleukin-1β (IL-1β), IL-6, IL-8, the p40 protein of IL-12, and the costimulatory molecules CD80 and CD86.
The types of cells that respond to CpG DNA—B cells, dendritic cells (DCs) and macrophages—are also stimulated by other pathogen-derived pattern-recognition factors, such as LPS. In general, the PRRs of the innate immune system are situated on the cell surface, where they are probably best able to detect microbes. Although cell-surface proteins that bind DNA are well described, and have been proposed to mediate immune activation by CpG motif (Liang H et al., J Clin Invest 98:1119-1129 (1998)), this binding is sequence-independent and does not bring about cell activation. Krieg A M et al., Nature 374:546-549 (1995); Yamamoto T et al., Microbiol Immunol 38:831-836 (1994); Häcker H et al., EMBO J. 17:6230-6240 (1998). Because CpG ODNs that have been immobilized to prevent cell uptake are nonstimulatory (Krieg A M et al., Nature 374:546-549 (1995); Manzel L et al., Antisense Nucleic Acid Drug Dev 9:459-464 (1999)), it appears that CpG ODN probably work by binding to an intracellular receptor. In support of this hypothesis, drugs such as chloroquine, which interfere with the endosomal acidification/processing of ODNs, specifically block the immune stimulatory effects of CpG DNA. Häcker H et al., EMBO J. 17:6230-6240 (1998); Macfarlane D E et al., J Immunol 160:1122-1131 (1998); Yi A K et al., J Immunol 160:4755-4761 (1998). It has been proposed that an endosomal step is required for the CpG-induced signal transduction pathways. Häcker H et al., EMBO J. 17:6230-6240 (1998); Yi A K et al., J Immunol 160:4755-4761 (1998). How the information contained in unmethylated CpG-motifs of bacterial DNA trigger changes in gene expression has not previously been discovered.
Since the receptor for bacterial DNA has been unknown, development of screening for optimal CpG motifs through direct binding analysis has been limited. An additional complication appears to be species-specific selectivity for CpG sequence, i.e., an optimal sequence for one species may not be optimal for another.